1. Field of Invention
This invention relates to the interpretation of data from an assay of a biological cell sample, and more particularly, to correction of DNA quantitative measurements in a tissue section.
2. Description of Prior Art
Cancer diagnosis and prognosis is largely dependent on the pathologic examination of tissue surgically removed from a patient. The specific diagnosis is made by a pathologist, who classifies the tumor by site and by cell of origin after examining stained histologic sections of the fixed, paraffin-embedded cancer tissue. The prognosis depends on many factors, including the specific diagnosis, the presence and pattern of tumor metastasis, the extent of tumor at its site of origin and its proximity to vital structures, and the tumor grade as assessed by a pathologist. In some organs, such as the prostate, the usual determinants of prognosis are inadequate to provide a patient-specific prognosis, especially when such is desired prior to definitive therapy. Consequently, other prognostic indicators have been sought.
One prognostic indicator which has been valuable in the cancers of certain organs is DNA ploidy, which is the ratio of the quantity of DNA in a cancer cell to that in a normal cell in the resting phase of its growth cycle. In general, tumors with normal resting-phase cellular DNA content (diploid) have a better prognosis than those with twice that amount (tetraploid), and these in turn have a better prognosis than those with abnormal DNA content which is not tetraploid (aneuploid).
The cellular DNA is located in the nucleus. Various methods have been developed for measuring the DNA content of whole nuclei. These methods do not make it possible for the measured cells to be correlated with their position or appearance in a standard histologic section. Thus, it is likely that normal cells will be measured together with tumor cells. Also, distinct areas of tumor cannot be measured separately. An even more important consideration is that very small samples, such as prostate thin core biopsies, are unsuitable.
All of these limitations have been overcome by measuring the DNA content of nuclei and partial nuclei in Feulgen-stained standard histologic sections. A new problem is created, however, by the inevitable inclusion of partial nuclei among the analyzed nuclei. In many sections, because the nuclear diameter exceeds the section thickness, all of the nuclei in the section will be partial. In U.S. Pat. No. 5,235,522 to Bacus for Method and Apparatus for Automated Analysis of Biological Specimens, an apparatus and method for measuring the DNA content of nuclei in tissue sections is described, as well as a method for correction of DNA measurements necessitated by the analysis of partial nuclei. Bacus and Bacus also have described this method of correcting DNA measurements in A Method of Correcting DNA Ploidy Measurements in Tissue Sections, published in Modern Pathology, Vol. 7, pp. 652-664, 1994. The Bacus correction makes three assumptions: 1) all nuclei are spherical, 2) all nuclei have a homogenous intranuclear DNA distribution, and 3) all nuclei with a profile area greater than .pi.T.sup.2 /4, where T is the section thickness, have been sectioned such that the center of the nucleus lies midway between the top and bottom of the tissue section. In an actual tissue section, the nuclei deviate from perfect spheres, the DNA distribution may not be homogeneous, and the nuclei are sectioned at essentially random positions and orientations. It is clear that the Bacus correction undercorrects most of the measurements, and would overcorrect some of the measurements, such as those made on a central section of an ellipsoidal nucleus aligned with the plane of the section, or those made on a centrally-sectioned nucleus in which most of the DNA is concentrated centrally. The Bacus method creates a histogram (FIG. 1) from the corrected DNA measurements, which shows a large number of bars 1 to the left of the bar representing the true whole-nucleus DNA content 2. These bars 1 represent partial nuclei for which the Bacus correction was insufficient, but may also be interpreted or misinterpreted as subpopulations of cells with different DNA content, or as cells at different points in the cell cycle. In many cases, such a misinterpretation might result in the classification of a prognostically favorable tumor as unfavorable. In cases of undercorrection or overcorrection of the modal DNA quantity, a tumor might be considered more or less prognostically favorable than would be inferred from its true ploidy. Also, histograms of quantitative DNA measurements in whole-nucleus preparations show very discreet peaks, reflecting discreet ploidy values of the measured nuclei; but when partial nuclei are measured, whether or not the measurements are corrected, the peaks are very blurred and may not be distinguishable as such (FIG. 1). The Bacus method allows the operator to define classes of attributes, thereby excluding many unwanted partial nuclei from analysis; such an approach is helpful but may be of limited value because the a priori classes may not accord with the natural classes in the specimen being analyzed.